Methods for characterizing glycoproteins and generating antibodies for same

ABSTRACT

The invention provides methods for preparing a peptide useful in generating an antibody specific for the glycosylated form of a glycopolypeptide wherein the glycosylated portion is isolated and a peptide corresponding to amino acids adjacent to a glycosylated N-linked glycosylation site is prepared.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/891,487, filedAug. 9, 2007, now U.S. Pat. No. 7,879,799, which claims the benefit ofpriority of United States Provisional Application No. 60/837,254, filedAug. 10, 2006, United States Provisional Application No. 60/876,883,filed Dec. 22, 2006, and United States Provisional Application No.60/878,753, filed Jan. 5, 2007. The content of each of these documentsis incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under grant number RO1-AI- 41109-01 awarded by the National Institutes of Health and grantnumber NO1-HV-28179 awarded by the National Heart, Lung and BloodInstitute. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of thesequence listing via the U.S.P.T.O EFS-WEB server, as authorized and setforth in MPEP §1730 II.B. 2(a)(C), is incorporated herein by referencein its entirety for all purposes. The sequence listing is identified onthe electronically filed text file as follows:

File Name Date of Creation Size (bytes) 655652001201Seqlist.txt Dec. 28,2010 23,275 bytes

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of proteomics andmore specifically to glycoproteins and membrane proteins.

The molecular composition and dynamic organization of the plasmamembrane (PM) determines how a cell can interact with its environment atany given moment in time. Proteins embedded in the membrane that haveexposed, extracellular domains are crucial for cell-cell communication,interaction with pathogens, binding of chemical messengers, and responseto environmental perturbations. In order to distinguish between proteinsthat localize to intracellular and plasma membrane domains, methods areneeded that will allow for the specific identification of the proteinspresent on the surface of any given cell.

Thus, there exists a need to efficiently identify, characterize andgenerate reagents for membrane proteins. The present invention satisfiesthis need and provides related advantages as well.

SUMMARY OF INVENTION

The invention provides methods for generating an antibody specific forthe deglycosylated form of a glycopolypeptide using a peptidecorresponding to an N-linked glycosylation site of a glycopolypeptide.The invention additionally provides methods for generating an antibodyspecific for a glycopolypeptide using a peptide corresponding to aminoacids adjacent to an authentic N-linked glycosylation site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary chemistry for the covalent modification ofcarbohydrate using the biotinylation reagent biocytin hydrazide. FIG. 1shows that covalent modification of carbohydrates (A) takes placefollowing oxidation to aldehydes (B), forming a covalent hydrazone bond(C). The biotinylation reagent, biocytin hydrazide (D) contains a longspacer chain and will not cross the plasma membrane.

FIG. 2 shows the results of a cell surface labeling technique that iscell surface specific. Shown are Ramos B cells upon visualization of thetagged cell surface proteins, which are subsequently identified viaLC-MS/MS.

FIG. 3 shows identified proteins of lipid raft co-isolated proteinsbefore and after applying the cell surface glyco-capture technology.FIG. 3A shows dentified proteins in a membrane preparation without usingthe cell surface glycocapture technology. FIG. 3B shows identifiedproteins in a membrane preparation using the cell surface glyco-capturetechnology.

FIG. 4 shows mass spectrometry results for a single glycoprotein. In theupper window (FIG. 4A) all peptides identified for Neogenin are shown(SEQ ID NOS:38-41). Grey residues mark the location of NXS/Tglycosylation motifs. The peptides are shown within the protein sequencebelow (FIG. 4B) (SEQ ID NOS:42-46, respectively in order of appearance),with the sites of glycosylation indicated in the shaded boxes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to efficiently identifying membraneproteins, in particular the extracellular domains of membraneglycoproteins. The invention further relates to reagents such asantibodies that bind to deglycosylated or native forms of aglycoprotein.

Cell surface proteins of human cells can be markers of disease and arepotential targets for pharmaceutical intervention. The present inventionrelates to a technology to selectively identify N-linked cell surfaceglycoproteins via mass-spectrometry. With this technique, proteins withN-linked carbohydrates that reside only on the extracellular cellmembrane at a given time are purified. Because the motifs ofglycosylation are known, and because the technique causes a mass shiftof one mass unit at the site of carbohydrate attachment, the methodreveals the exact site of N-glycosylation within an identified trypticpeptide of a cell surface glycoprotein. Given that carbohydrates arehighly hydrated and hydrophilic, the mass spectrometry findings ofmethods of the invention permit inferences about the structuralorientation of the protein in the membrane. Specifically, the resultsidentify regions of the protein sequence that are (1) oriented towardsthe extracellular environment as opposed to being buried within thehydrophobic core of the protein and (2) located on the outer side of thecell membrane.

The results disclosed herein using methods of the invention haveprofound implications for research which require specific antibodiesagainst cell surface and secreted proteins. Historically, raisingantibodies against human cell surface proteins has been difficult forseveral reasons. First, there is a high degree of sequence similaritybetween proteins in different species which causes tolerance toimmunization. Second, it is difficult to select immunogenic peptidesfrom cell surface proteins because they are so frequently modified bycarbohydrates and other molecular entities that conceal the epitope inthe native protein from any antibody generated using a syntheticpeptide.

To date, immunizing peptides are chosen according to a combination oftheoretical parameters and “common knowledge”. Most frequently, apeptide is chosen from the amino or carboxy terminus of the cell surfaceprotein, assuming that this location in the sequence string will make itaccessible in the native protein. This algorithm is a crude science; itis usually made without knowledge of the orientation of the proteinwithin the membrane, or by relying on theoretical orientation predictionalgorithms. The relative antigenicity of a peptide string can beestimated to some extent by using prediction algorithms, though again,these programs do not take orientation into account.

As disclosed herein, methods are provided for the selection of peptidesfrom cell surface proteins for the generation of antibodies based on theprevious MS identification of N-glycosylation sites that shoulddramatically increase the likelihood of developing a reagent thatrecognizes the native protein compared to standard methods.

The identification of disease specific biomarkers is one of the majorgoals of translational research. “Biomarkers” such as prostate specificantigen (PSA), are broadly defined as an assayable characteristic thatcorrelates with a biological process. Protein biomarkers have recentlyreemerged as prime research targets because (1) they can be sampledeasily from body fluids, (2) they are reflective of host physiology, (3)they have demonstrated value as diagnostic agents for a number ofclinical conditions, and (4) protein profiling technology and improvedproteomics techniques have become feasible. Protein biomarkers in cancercan generally be classified as those that (1) predict the tumor behaviorbased on the presence or absence of the marker within the tumor sample(local biomarkers) and (2) those that, because of their presence in bodyfluid, can be used to screen for or monitor disease (disseminatedbiomarkers). These groups are not necessarily mutually exclusive. Thefirst category of biomarkers includes proteins such as the estrogenreceptor and Her-2/neu protein in breast cancer. Pathologic analysis nowroutinely includes these markers because of their relationship toprognosis and response to therapy. The latter class of biomarkersincludes PSA and alpha fetoprotein, which can be used either as ascreening test or a marker for disease progression or recurrence (forprostate cancer or germ cell tumor, respectively) (Kolonel et al.,Cancer Epidemiol. Biomarkers Prev. 9 795-804 (2000)).

The development of new research tools in the last decade has fosterednew approaches to identify biomarkers of both classes. Research toidentify the local protein biomarkers that can differentiate tumorbehavior has been performed in earnest for many years through extensiveanalysis at both the DNA and protein levels using both cell lines andtumor samples. There have been exciting breakthroughs recently ingenomic analysis using array platforms (reviewed in Ntzani andIoannidis, The Lancet 362:1439-1444 (2003)). The field of proteomics hasalso developed dramatically over the last decade, allowing in-depthstudy of cancer tissues and cell lines using high-throughput massspectrometry based techniques (Aebersold and Mann, Nature, 422:198-207(2003); Domon and Aebersold, Science, 312:212-217 (2006)). Whilegenerating numerous insights into molecular function and systemsbiology, the translation of specific protein identity into assays withclinical utility has lagged. Interest in biomarkers persists despitelimited successes because effective cancer biomarkers could dramaticallyimpact health and research through early disease detection, guidingtreatment through “personalized” treatment plans, and enhancing drugdevelopment by indicating therapeutic responses. Unfortunately, few newtests based on proteomic or genomic technologies are forthcoming; newdrug and biologic submissions to the FDA have dropped over the lastdecade, despite hopes raised with the human genome project (Anderson andHunter, Mol. Cell Proteomics, 5:573-588 (2006)).

One of the major reasons for the slow translation of genomic andproteomic insights into new diagnostic assays are the difficulties ingenerating suitable test platforms. In the case of proteomics, massspectrometry serves well as a research instrument; however the cost andcomplexity have limited integration into the clinical realm. Inaddition, validating clinical biomarkers typically requires sensitiveand accurate quantification of candidates in hundreds to thousands ofindividual samples. The current gold standard of quantitative proteinbioassay is the Enzyme-Linked Immunosorbent Assay (ELISA). A goodperforming ELISA can be run at high-throughput with both extraordinarysensitivity (detection limits ˜5 pg/ml) and specificity. Antibody basedreagents are also the gold standard in immunohistochemistry, where onlya single antibody is required for pathologic analysis of a clinicalsample. Such reagents are indispensable in providing certainty forcomplex medical diagnosis and in tailoring treatment. Numerous examplesexist of antibody based reagents. Screening with a PSA ELISA, whilesomewhat controversial (Harris and Lohr, Ann. Intern. Med. 137:917-929(2002); Ann. Intern. Med. 137:915-916 (2002); Carroll et al., Urology57:217-224 (2001); Smith et al., CA Cancer J. Clin. 50:34-49 (2000)), iswidely practiced (Routh and Leibovich, Mayo Clin. Proc. 80:899-907(2005)). Antibodies form the basis of numerous other serum assays in theevaluation of malignancy including alpha fetoprotein and βhCG in germcell tumors and carcinoembryonic antigen (CEA) in colonic neoplasms;antibody based flow cytometry is standard in the diagnosis ofhematologic malignancies. Immunotherapy has been demonstrated to behighly efficacious in the treatment of malignancies; Trastuzumab (breastcancer), rituximab (B-cell lymphoma), bevacizumab and cetuximab (Coloncancer), Alemtuzumab (chronic lymphocytic leukemia) are a few examplesof agents widely used in clinical oncology (Armitage et al., ClinicalOncology, 3rd edition, New York: Churchill Livingstone (2004)). Numerousother antibody based therapeutics are currently under investigation inmultiple areas of clinical medicine.

ELISAs are costly to develop (≧$40,000 per biomarker) and have a longdevelopment lead-time (>1 year). Much of the difficulties associatedwith producing new immunologic reagents lie in generating a specificB-cell immune response. To add to this challenge, for clinical tests itis highly desirable to have immunoreactivity proteins in the nativestate. While some assays such as western blotting do not require suchreactivity, it is the sine qua non for flow cytometric cell sorting,pathologic analysis, ELISA analysis, and the like. In addition, in vivotherapeutic assays or methods require antibodies that react withproteins in the native state. Immunization of an animal for the purposeof generating an antibody must provide the host B-cell with an antigenthat is unique to the protein of interest and in a state of presentationthat is identical to that of the desired target. Most B-cell epitopesare composed of different parts of the polypeptide chain that arebrought into spatial proximity by protein folding, which is referred toas “discontinuous” epitopes. It has been estimated that forapproximately 10% of epitopes, the corresponding antibodies cross-reactwith a continuous linear peptide fragment (Pellequer et al., MethodsEnzymol. 203:176-201 (1991); Barlow et al., Nature, 322:747-748 (1986));such epitopes are denoted linear or continuous epitopes.

Antibodies can be generated through inoculation of whole protein or apeptide epitope. Whole protein is frequently obtained as a recombinantfusion protein produced in bacteria due to the ease of molecular biologyrequired and the abundance of expression and purification systems on themarket. Whole protein exposes the host animal to both continuous anddiscontinuous epitopes. Unfortunately, recombinant proteins arefrequently inadequate immunogens; they are often incorrectly folded withmisaligned disulfide bonding. In addition, membrane proteins are noteasily expressed in bacterial or eukaryotic systems. Post translationalmodification within the expression system often does not recapitulatethat of the original organism; glycosylation is absent in prokaryoticsystems and is not guaranteed to be identical in eukaryotic systems, forexample, insect cells or yeast cells. Finally, purification frequentlygenerates impure mixtures of proteins, complicating the antibodyresponse following immunization.

A second approach commonly utilized to generate antibodies is toimmunize an animal with a continuous peptide sequence from the proteinof interest. Small molecules such as peptides are not usuallyimmunogenic, even when co-administered with adjuvants to increase theintensity of the immune response. In order to generate a response topeptide immunogens, they are generally attached to an immunogeniccarrier protein, termed a hapten. The most commonly used carrier proteinis keyhole limpet hemocyanin (KLH), a mollusk respiratory heme protein.Because KLH shares no homology with vertebrate proteins, antibodyresponses against the carrier protein results in no cross-reactivity inassays against mammalian proteins. A second option frequently used isthe Multiple Antigen Peptide System (MAPS) in which multiple identicalpeptides linked to a small immunogenically inert branched lysine core(Tam, Proc. Natl. Acad. Sci. USA. 85:5409-5413 (1988); Tam and Zavala,J. Immunol. Methods 124:53-61 (1989)). This MAP molecule is a largerthree dimensional molecule that does not need a carrier protein toinduce an antibody response; MAPS peptides avoid generation ofantibodies to the commonly used carrier proteins, although they maycontain antibodies specific to the MAPS core structure. Altogetherpeptide immunization is a highly inexact science that depends on bothknowledge and chance.

The greatest difficulty in using a continuous peptide antigen ischoosing a sequence that produces an antibody to the full length foldedprotein. Extensive research over the last three decades has producedlittle progress in predicting linear B-cell epitopes. The classicalmethods employed use a variety of propensity scales to assign anumerical value to every amino acid based on studies of theirphysico-chemical properties. These algorithms then apply a runningaverage window to generate a local average score. The first score wasthat of Hopp and Woods (Hopp, Pept. Res. 6:183-190 (1993); Hopp andWoods, Proc. Natl. Acad. Sci. USA 78:3824-3828 (1981)), who developed ascale of hydrophilicity. Kyte and Doolittle proposed a similar methodbased on a separate scale of hydrophobicity (Kyte and Doolittle, J. Mol.Biol. 157:105-132 (1982)). A scale of peptide flexibility was firstintroduced by Karplus (Karplus and Schultz, Naturwissenschaften72:212-213 (1985)); a surface accessibility scale was also introduced(Emini et al., J. Virol. 55:836-839 (1985)). It has been shown thatprotein regions corresponding to the antigenic peptides are usuallyhighly mobile (Westhof et al., Nature 311:123-126 (1984); Tainer et al.,Nature, 312:127-134 (1984)). The number of available scales has grown toover 500 since the 1980's; most are subtle variants on those describedabove. Most of the propensity scales were developed through training onproteins for which extensive epitope mapping was carried out; generallythese datasets were small and the accuracy of the methods unknown.Recently, Blythe and Flower benchmarked the performance of 484 of thepropensity scales on a large data set (50 proteins) with extensiveepitope mapping and found that even the best methods predict onlymarginally better than a random prediction (Blythe and Flower, ProteinSci. 14:246-248 (2005)).

A number of efforts have attempted to combine various propensity scalesto improve predictive power. Alix developed a program called PEOPLE,which predicts the location of linear B-cell epitopes using combinationsof propensity scales including secondary structure, hydrophilicity,surface accessibility, and flexibility to generate an “antigenic index”(Alix, Vaccine 18:311-314 (1999)). Odorico developed a program,BEPITOPE, for predicting the location of linear B-cell epitopes using acombination of hydrophilicity, accessibility, and turns in proteinspropensity scales (Odorico and Pellequer, J. Mol. Recognit. 16:20-22(2003)). Sollner and Mayer developed a machine learning method topredict B-cell epitopes by combining propensity scales, reinterpretingsome propensity scales (substituting pair wise difference betweenadjacent amino acids rather than moving averages), and using residuefrequencies such as di and tripeptide occurrences within a local peptideneighborhood (Sollner and Mayer, J. Mol. Recognit. 19:200-208 (2006)).The authors claim improvement in performance over other predictionmodels. Another recent paper reports that a hidden Markov model combinedwith a hydrophilicity scale improves epitope prediction called BepiPred(Larsen et al., Immunome Res. 2:2 (2006). It is not clear at this timehow much of an improvement these newer methods actually provide; nostudy has been done to benchmark the performance of any of thesealgorithms.

The consensus approach recommended by most commercial entities producingcustom polyclonal and monoclonal antibodies is to empirically choose theamino or carboxy termini of the protein of interest as these sequencesare more frequently exposed on the exterior of the protein. Indeed manyof the commercially available antibodies available for sale are raisedagainst these epitopes. An informal survey of the catalogs of a numberof large distributors of antibodies indicates that approximately 40% ofall the antibodies for sale are directed at the amino or carboxytermini. The guidance provided by most of these vendors reveals thatbeyond the empiric amino and carboxy terminal strategies, therecommendation is to use one of the propensity scales introduced morethan 20 years ago. Most providers provide “custom” design of epitopes,although this generally uses propensity scales and a limited number ofrules of thumb.

There is no body of work or database that indicates how well an antibodygenerated by a conjugated peptide will perform. The best source ofinformation is the antibody producers themselves, who frequently performcustom antibody synthesis for clients as well as develop specificantibodies for sale as individual reagents. Six large producers ofcustom antibodies were surveyed, and their responses to queriesregarding success rate was generally in agreement, providing the bestestimates, summarized as follows. There is a very high success rate ingenerating a high titer antibody to a conjugated peptide epitope. Thisis determined by performing an indirect ELISA against the immunizingpeptide alone. A number of vendors guarantee such a response, though the“best guess” from those consulted is 90-95% success. Most failures canbe addressed by re-conjugation and immunization of a new animal orchanging to a different animal type. The experts also generally agreethat the range of success for these antibodies in reacting to wholeprotein in a western blot (where it has been denatured), isapproximately 50% with a range from 40 to 60% being quoted. There wasmuch more uncertainty regarding the success of antibodies in assaysrequiring specificity to native proteins, estimated to be about 30 to40%. This informal survey highlights one of the deficiencies in thefield; no publicly available study defines these parameters with anyconfidence. In addition, the low numbers associated with the successrate for applications directed at native epitopes means that significantresources are wasted in the process.

It is clear that the state of the art for choosing peptide antigens isunsatisfactory. The deficiencies in this field are made more apparentwhen taken in the context of the developments of the last decade. Arraybased platforms interrogating tens of thousands of gene products cangenerate numerous biological insights that could be tested at theprotein level if reagents were available. Proteomic platforms likewiseproduce hundreds to thousands of observations in the research settingthat need follow up studies on clinical specimens. To add to thefrustration with the current situation, there have been pathologicplatform developments such as tissue microarrays that allow the rapidassessment of hundreds or even thousands of patient samples withantibodies for markers of interest. Unfortunately if no antibody isavailable for a protein, these platforms are moot. It is also clear thatthe industry for generating both polyclonal and monoclonal antibodies ismature and ready to handle the demand that would come if generation of areactive antibody was more certain. There are hundreds of vendorsnationally using rabbits, mice and chickens for polyclonal production.Monoclonal production is now possible with mouse, rat, and rabbit Bcells; techniques have recently been commercialized to generatemonoclonal antibodies through the use of a recombinant retrovirus thatconstitutively expresses the v-abl and c-myc oncogenes. When used toinfect immunized mice, this virus rapidly and efficiently inducesplasmacytomas to secrete antigen specific monoclonal antibodies (Huppiet al., Biotechniques, 29:1100-1106 (2000); Largaespada et al., J.Immunol. Methods, 197:85-95 (1996)).

In the future, antibodies are likely to play a growing role in cancerdiagnostics and therapeutics as findings from new high throughputtechnologies define novel protein targets. The methods of producingantibodies, both polyclonal and monoclonal have reached maturity.Unfortunately, guidance on the choice of a peptide epitope has notchanged in over two decades; empiric rules and propensity scales stilldominate most prediction schemes. The end result is that at least halfof conjugated peptide immunizations produce no antibody capable ofreactivity, even in a denaturing immunoblot. When it comes to moredemanding tasks such as immunohistochemistry, immunoprecipitation andfluorescence activated cell sorting (FACS), the performance issignificantly worse. This represents a tremendous waste of time andresources that demands a better solution. As disclosed herein,proteomics based methods have been developed that identify glycosylationsites on soluble and membrane proteins. Because the carbohydrate groupsare hydrophilic and on the protein surface, the methods disclosed hereinprovide a strategy focused on this protein neighborhood, also referredto herein as the glyprox strategy, that will provide more efficientproduction of antibodies against glycoproteins than the historicalmethods.

As disclosed herein, two proteomic strategies have been developed toanalyze biological samples based on the selective capture ofglycosylated proteins using hydrazide chemistry (Zhang et al., Nat.Biotechnol. 21:660-666 (2003)) that improves the effectiveness ofgenerating antibodies to cell surface and secreted proteins. The initialmotivation for selecting glycoproteins was two fold: (1) to use thisselectivity to reduce the complexity and dynamic range of serum becausealbumin is lightly glycosylated and (2) to specifically capture proteinsdestined for the cell membrane and secretion. The methods relate tobiomarker identification using a glyco-specific methodology. A methodfor capturing glycoproteins from solution is described in Example III.Methods for capturing membrane glycoproteins is described in Examples IIand IV. Example II details principles of membrane purification using amembrane impermeable bifunctional reagent, biocytin hydrazide. Anexemplary protocol for capturing cell surface glycoproteins is describedin Example I. It is understood that various modifications of theprotocol can be used, as understood by those skilled in the art, can beused so long as sufficient glycoproteins are analyzed, as desired.Studies demonstrating the efficiency of the technique for selectivepurification of membrane proteins from RAMOS and Jurkat lymphocyte celllines are described in Example II. The development of a list of T celloverrepresented proteins is given in Example IV. Collectively theseresults indicate that proteomic analysis can be used as a highlyselective method for the identification of biomarkers such as cancerderived biomarkers that will serve as the basis for choosing targets forthe generation of antibodies.

In one embodiment, the invention relates to methods for generatingantibodies against enzymatically deglycosylated membrane glycopeptides.The methods relate to the generation of antibodies against theenzymatically deglycosylated forms of bona fide cell surfaceglycopeptides, generally plus and/or minus twenty five amino acidresidues from the glycosylation site, depending on antigenicitypredictions, that have been previously identified by mass spectrometry.This method is based on the fact that enzymatic deglycosylation of anN-linked glycopeptide with Peptide-N-Glycosidase F (PNGaseF) convertsthe asparagine residue to its acidic form, aspartic acid. The antibodyis raised against a peptide string in which the asparagine residue isdeliberately changed to aspartic acid. The newly generated antibody isable to detect a protein target after enzymatic deglycosylation withPNGaseF to unmask the epitope. This technique has wide potentialapplications including but not limited to western blotting,immunohistochemistry, and cell sorting. Although exemplified withPNGaseF, it is understood that other glycosidases, includingN-glycosidases and O-glycosidases that can be used to cleave N-linkedand O-linked carbohydrate moieties, respectively.

There are three aspects of this method of the invention. First, usingthe site of glycosylation indicates that the target peptide sequence ispresent on the exterior of the protein. Second, raising an antibody to apeptide “mutated” by conversion of asparagine to aspartic acid has ahigher likelihood to induce an immune response as it unmasks a typically“invisible” sequence which itself deviates from evolutionarily conserved“self”. The invisibility derives from the fact that the attachedcarbohydrate molecules cause steric hindrance, preventing antibodiesaccess to this residue. Third, aspartic acid is known have greaterantigenicity than asparagines. These three features together willsignificantly increase the likelihood of generating a useful antibodyagainst a protein of interest.

In another embodiment, the invention provides methods for generatingantibodies against sequences adjacent to established sites ofglycosylation. The method relates to generating antibodies usingimmunizing sequences based on close proximity to a glycosylation sitesuch as an N-glycosylation site established as an authenticglycosylation site using mass spectrometry. Close proximity is definedas being within twenty-five residues up or downstream of the site ofglycosylation. Actual sequence for immunization will be chosen dependingon predicted antigenicity. Like the method discussed above, the targetsagainst which the antibodies are raised are known to be on the portionof the protein exterior to the membrane. Also, immunizing peptides willinclude portions of the sequence identified by mass spectrometry, whichmeans that these residues were not modified post-translationally, andwill be accessible to antibodies. Using the mass spectrometry basedidentification method, the peptides that are identified have unmodifiedamino acids surrounding the glycosylation site. Any changes in theweight of the amino acid, such as methylation, phosphorylation, or otherpost-translational modifications, which change the weight of thepeptide, will prevent the identification. Thus the observation of adeglycosylated peptide means that all amino acids are unmodified. Thisalso means that this is a good sequence for raising an antibody since,as in the native protein, the amino acids are unmodified. If one usessequences outside of those identified, there is a risk that there is anunexpected modification on an amino acid which will make it differentfrom the synthetic sequence used for immunization. This portion ofidentified peptide provides some reassurance that part of the immunizingsequence is just as it is in the native protein.

One of the most significant advantages to this method is that theknowledge of sites of glycosylation provides significant insight intothe local structure of the protein. Targeting the region around theglycosylation site dramatically increases the chance that an antibodyraised against a peptide epitope will be reactive toward a nativeprotein. This strategy should work especially for generating antibodiesfor FACS and ELISA assays as well any biopharmaceutical agent, whichmust be reactive toward native proteins.

Protein glycosylation has long been recognized as a very commonpost-translational modification. Carbohydrates are linked to serine orthreonine residues (O-linked glycosylation) or to asparagine residues(N-linked glycosylation) (Varki et al. Essentials of Glycobiology ColdSpring Harbor Laboratory (1999)). Protein glycosylation, and inparticular N-linked glycosylation, is prevalent in proteins destined forextracellular environments (Roth, Chem. Rev. 102:285-303 (2002)). Theseinclude proteins on the extracellular side of the plasma membrane,secreted proteins, and proteins contained in body fluids, for example,blood, serum, plasma, cerebrospinal fluid, urine, breast milk, saliva,lung lavage fluid, pancreatic juice, semen, and the like. These alsohappen to be the proteins in the human body that are most easilyaccessible for diagnostic and therapeutic purposes.

Due to the ready accessibility of body fluids exposed to theextracellular surface of cells and the presence of secreted proteins inthese fluids, many clinical biomarkers and therapeutic targets areglycoproteins. These include Her2/neu in breast cancer, human chorionicgonadotropin and α-fetoprotein in germ cell tumors, prostate-specificantigen in prostate cancer, and CA125 in ovarian cancer. The Her2/neureceptor is also the target for a successful immunotherapy of breastcancer using the humanized monoclonal antibody HERCEPTIN™ (Trastuzumab)(Shepard et al., J. Clin. Immunol. 11:117-127 (1991)). In addition,changes in the extent of glycosylation and the carbohydrate structure ofproteins on the cell surface and in body fluids have been shown tocorrelate with cancer and other disease states, highlighting theclinical importance of this modification as an indicator or effector ofpathologic mechanisms (Durand and Seta, Clin. Chem. 46:795-805 (2000);Freeze, Glycobiology 11:129R-143R (2001); Spiro, Glycobiology 12:43R-56R(2002)). Therefore, a method for the systematic and quantitativeanalysis of glycoproteins would be of significance for the detection ofnew potential diagnostic markers and therapeutic targets, and themethods of the invention for generation of reagents such as antibodiesare particularly useful for generating potential antibody therapeuticagents.

One of the major biosynthetic functions of the endoplasmic reticulum isthe covalent addition of sugars to proteins; most of the soluble andmembrane-bound proteins that are made in the ER are glycoproteins(Alberts et al., Molecular Biology of the Cell New York: Garland Science(2001)). Glycosylation in the ER consists of an en bloc transfer of apreformed precursor oligosaccharide to the side-chain amino group of anasparagine residue in the protein (N-linked). The preformedoligosaccharide is transferred to the target asparagine in a single stepimmediately after that amino acid translocates into the ER lumen.N-linked glycosylation sites generally fall into the N—X—S/T sequencemotif in which X denotes any amino acid except proline (Bause, Biochem.J. 209:331-336 (1983)). A second type of glycosylation event in theGolgi links oligosaccharides to the hydroxyl group on the side chain ofa serine, threonine, or hydroxylysine, called O-linked glycosylation.

The function of N-linked glycosylation is unknown. It is known that someproteins require N-linked glycosylation for proper folding (Daniels etal., Mol. Cell, 11:79-90 (2003); Petrescu et al., Biochemistry,39:5229-5237 (2000)). Two ER chaperone proteins, calnexin andcaireticulin, bind to oligosaccharides on incompletely folded proteinsand retain them in the ER. Another possible mechanism arises becausehydrophilic sugar chains have limited flexibility and protrude from theprotein surface. This can thus limit the approach of othermacromolecules which tends to make a glycoprotein more resistant toproteolytic attack. Experiments have shown that in some cases,glycosylation is not an absolute requirement for proper folding, but theunglycosylated protein degrades quickly. Finally glycosylation may beinvolved cell-cell adhesion (Lasky, Annu. Rev. Biochem. 64:113-139(1995)) and molecular recognition (Lis and Sharon, Chem. Rev. 98:637-674(1998)).

A recent large scale analysis was performed characterizing the sites ofprotein glycosylation using a large crystallographic database including386 sites of N-linked carbohydrates (Petrescu et al., Glycobiology,14:103-114 (2004)). The sites of glycosylation were characterized asexposed convex surfaces 33%, deep recesses 10% and on the edge ofgrooves with the glycan filling the cleft 20%. These authors also foundan elevated probability of finding glycosylation at sites wheresecondary structure changes. A third observation was that there weredeviations in the expected amino acid composition surrounding theglycosylation sites, particularly an increased occurrence of aromaticresidues before the asparagine and threonine. The authors proposed thatglycans may play a role in organizing the folding process and that thepositions of glycosylation sites may have evolved to act as landmarksfor ending or starting regions of regular secondary structure to promoteefficient folding. In addition the local hydrophobicity supports thehypothesis that glycans are involved in covering/stabilizing hydrophobicpatches of the protein surface (Toyoda et al., J. Biochem. 131:511-515(2002)).

In one embodiment, the invention provides a method for generating anantibody specific for the deglycosylated form of a glycopolypeptide. Themethod can include the steps of generating a peptide corresponding to anN-linked glycosylation site of a glycopolypeptide, wherein theasparagine corresponding to the N-linked glycosylation site is mutatedto an aspartic acid; and generating an antibody having specific bindingactivity for the peptide, wherein the antibody has specific bindingactivity for the deglycosylated form of the glycopolypeptide. Asdisclosed herein, the cleavage of an N-linked glycosylation site withPNGase-F cleaves between the innermost N-acetylglucosamine (GlcNAc) andasparagine residues of high mannose, hybrid and complexoligosaccharides. This cleavage deaminates the asparagine residue to anaspartic acid residue. As used herein, a “mutated” polypeptide sequencerefers to a sequence in which one or more amino acid residues areindependently substituted with another amino acid, such as aspartic acidfor asparagine, relative to a parent sequence, for example, relative toa naturally occurring sequence. By mutating the asparagine glycosylationsite to an aspartic acid, the peptide used to generate an antibodyagainst the glycopolypeptide represents the deglycosylated from of theglycopolypeptide. Antibodies generated against such peptides thereforecan bind to the deglycosylated form of the glycopolypeptide and can beused, for example, to detect binding to the deglycosylated form.

Generally when making anti-peptide antibodies, it is desirable to usepeptides of a minimum size of 6 amino acids (see Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press(1988); Doolittle, Of URFS and ORFS: A Primer on How to Analyze DerivedAmino Acid Sequences, University Science Books, Chapter 5, pp. 63-79(1986). In one embodiment, the peptide used comprises independently atmost 3 amino acids on the amino and carboxyl terminal side of theN-glycosylation site. As used herein, “independently” means that thenumber of amino acids on the amino or carboxyl terminal side of theN-glycosylation site are independently chosen to be the recited size anddo not necessarily have to be the same size on each side of theglycosylation site. For example, a peptide comprising independently atmost 3 amino acids on the N-terminal and C-terminal side can be selectedwith 2 amino acids on the N-terminal side and 3 amino acids on theC-terminal side. Such an exemplary peptide independently comprising atmost 3 amino acids on the N-terminal and C-terminal side of theN-glycosylation site would contain 6 amino acids, the aspartic acid atthe mutated glycosylation site, 2 N-terminal amino acids and 3C-terminal amino acids flanking the glycosylation site. Other exemplarypeptides independently comprising at most 3 amino acids on theN-terminal and C-terminal side of the N-glycosylation site would be apeptide containing 3 N-terminal amino acids and 2 C-terminal aminoacids, or a peptide containing 3 N-terminal amino acids and 3 C-terminalamino acids. In another embodiment, the peptide can compriseindependently at most 5 amino acids, at most 6 amino acids, at most 7amino acids, at most 8 amino acids, at most 9 amino acids, at most 10amino acids, at most 11 amino acids, at most 12 amino acids, at most 13amino acids, at most 14 amino acids, at most 15 amino acids, at most 16amino acids, at most 17 amino acids, at most 18 amino acids, at most 19amino acids, at most 20 amino acids, at most 21 amino acids, at most 22amino acids, at most 23 amino acids, at most 24 amino acids, at most 25amino acids, at most 26 amino acids, at most 27 amino acids, at most 28amino acids, at most 29 amino acids, or at most 30 amino acids on theamino and carboxyl terminal side of the N-glycosylation site. It isunderstood that longer peptides can be synthesized as desired for aparticular use.

In another embodiment of a method for generating an antibody specificfor the deglycosylated form of a glycopolypeptide, the method canfurther comprise identifying the N-linked glycosylation site. Forexample, an N-linked glycosylation site can be identified by massspectrometry, as disclosed herein.

The invention additionally provides a method for generating an antibodyspecific for a glycopolypeptide. The method can include the steps ofgenerating a peptide corresponding to amino acids adjacent to anN-linked glycosylation site of a glycopolypeptide, wherein the N-linkedglycosylation site has been identified using mass spectrometry as anauthentic glycosylation site; and generating an antibody having specificbinding activity for the peptide, wherein the antibody has specificbinding activity for the native form of the glycopolypeptide. The nativeform is the folded form of the polypeptide, that is, non-denatured.

As used herein, an “authentic glycosylation site” refers to aglycosylation site that has been empirically and experimentallydetermined to be a bona fide glycosylation site. An authenticglycosylation site, having been experimentally determined to be anactual glycosylation site, is distinct from a putative glycosylationsite which has not been experimentally determined to be a glycosylationsite. As discussed herein, the consensus sequence for N-linkedglycosylation is N—X—S/T, and any given protein will generally have anumber of such consensus sequences which, if located on a secretedprotein or the extracellular domain of a membrane protein, are potentialN-linked glycosylation sites. However, it is known that not all suchsites are actually glycosylated at the consensus N—X—S/T site, and ithas been traditionally difficult to routinely determine which, if any,of such sites are actually glycosylated in a particular cell or tissue.However, as disclosed herein, methods of labeling glycopolypeptides andanalysis by mass spectrometry have allowed the identification ofauthentic glycosylation sites of glycopolypeptides. The generation ofantibodies to peptides adjacent to authentic glycosylation sitesincreases the likelihood that the antibodies will react with nativeprotein since the glycosylation sites are exposed and not buried withinthe folded glycopolypeptide.

As used herein, the phrase “adjacent to an N-linked glycosylation site”refers to the amino acids proximal to the glycosylation site in thelinear sequence. Amino acids adjacent to an N-linked glycosylation siteinclude the amino acids immediately adjacent to the glycosylation site,that is −1 amino acid on the N-terminal side and +1 amino acid on theC-terminal side. An adjacent amino acid can also include within up to afew amino acids N-terminal or C-terminal of the glycosylation site, forexample, −2 or +2, −3 or +3, −4 or +4, −5 or +5, and the like, so longas the peptide generated corresponds to amino acids adjacent to anN-linked glycosylation site such that an antibody generated against thepeptide has specific binding activity for the native form of theglycopolypeptide.

As discussed above, it is generally desired to use at least a 6 aminoacid sequence to generate an anti-peptide antibody. In a method forgenerating an antibody specific for an authentic N-linked glycosylationsite of a glycopolypeptide, the peptide can comprise, for example, atmost 6 amino acids on the amino or carboxyl terminal side of theN-glycosylation site. In addition, a peptide can comprise at most 7amino acids, at most 8 amino acids, at most 9 amino acids, at most 10amino acids, at most 11 amino acids, at most 12 amino acids, at most 13amino acids, at most 14 amino acids, at most 15 amino acids, at most 16amino acids, at most 17 amino acids, at most 18 amino acids, at most 19amino acids, at most 20 amino acids, at most 21 amino acids, at most 22amino acids, at most 23 amino acids, at most 24 amino acids, or at most25 amino acids on the amino or carboxyl terminal side of theN-glycosylation site. Larger peptides having at most 30, 35, 40, or moreamino acids can be used, as desired.

In one embodiment of a method of the invention, the glycopolypeptide canbeen isolated using hydrazide chemistry, for example, using biocytinhydrazide. Exemplary methods for isolating a glycopolypeptide usinghydrazide chemistry are described in the Examples.

Methods for identifying authentic glycosylation sites using massspectrometry are described in the Examples. It is understood that massspectrometry can be used to identify an authentic glycosylation site.However, when referring to the peptides comprising independently at mosta given number of amino acids or to those adjacent to a glycosylationsite such as a N-linked glycosylation site, it is understood that it isthe site of glycosylation that is identified by mass spectrometry, notnecessarily the entire peptide being used in the methods of theinvention. For example, mass spectrometry can be used to identify theglycosylation site and, based on the sequence of the peptide analyzed bymass spectrometry, the information can be used to identify theglycoprotein and the glycosylation site (see Example II). Theidentification of the glycoprotein is determined by comparing thesequence to a database of protein sequences and identifying the proteinthat corresponds to the peptide analyzed by mass spectrometry. Once theglycoprotein is identified, the known sequence of the glycoprotein,irrespective of the specific peptide analyzed by mass spectrometry toidentify the glycosylation site, can be used to select a desired peptidesize for use in a method of the invention, as disclosed herein. AlthoughMS methods are particularly useful for identifying authenticglycosylation sites, as disclosed herein, it is understood that othermethods to identify an authentic glycosylation site can also be used, asdesired.

Methods of mass spectrometry analysis are well known to those skilled inthe art (see, for example, Yates, J. Mass Spect. 33:1-19 (1998); Kinterand Sherman, Protein Sequencing and Identification Using Tandem MassSpectrometry, John Wiley & Sons, New York (2000); Aebersold andGoodlett, Chem. Rev. 101:269-295 (2001)). For high resolutionpolypeptide fragment separation, liquid chromatography ESI-MS/MS orautomated LC-MS/MS, which utilizes capillary reverse phasechromatography as the separation method, can be used (Yates et al.,Methods Mol. Biol. 112:553-569 (1999)). Data dependent collision-induceddissociation (CID) with dynamic exclusion can also be used as the massspectrometric method (Goodlett et al., Anal. Chem. 72:1112-1118 (2000)).

Methods for synthesizing peptides are well known to those skilled in theart. Methods for peptide synthesis and the production of peptidelibraries have been described (see, for example, Merrifield, MethodsEnzymol. 289:3-13 (1997); Fodor et al., Science 251:767 (1991); Gallopet al., J. Med. Chem. 37:1233-1251 (1994); Gordon et al., J. Med. Chem.37:1385-1401 (1994)).

Although exemplified herein with N-linked glycosylation, it isunderstood that methods of the invention can also be used with othertypes of authentically identified glycosylation sites, such as O-linkedglycosylation sites or other identified glycosylation sites, forexample, glycosylphosphatidylinisotol (GPI) anchored proteins. Forexample, peptides adjacent to authentic O-linked or GPI-linked sites canbe synthesized and antibodies generated, as described herein.

The invention additionally provide antibodies generated by the methodsdisclosed herein, for example, antibodies specific for thedeglycosylated form of a glycopolypeptide. It is understood that such anantibody binds specifically to the deglycosylated form of aglycopolypeptide but not the glycosylated form. The invention furtherprovides antibodies specific for a peptide corresponding to amino acidsadjacent to an authentic N-linked glycosylation site. Methods forpreparing antibodies are well known to those skilled in the art. As usedherein, the term “antibody” is used in its broadest sense to includepolyclonal and monoclonal antibodies, as well as antigen bindingfragments of such antibodies. An antibody useful in the invention, orantigen binding fragment of such an antibody, is characterized by havingspecific binding activity for an antigen or epitope, such as apolypeptide or a peptide portion thereof, of at least about 1×10⁵ M⁻¹.Thus, Fab, F(ab′)₂, Fd, Fv, single chain Fv (scFv) fragments of anantibody and the like, which retain specific binding activity for anantigen or epitope such as a polypeptide, are included within thedefinition of an antibody. Specific binding activity of an antibody foran antigen or epitope such as a polypeptide can be readily determined byone skilled in the art, for example, by comparing the binding activityof an antibody to a particular antigen or epitope such as a polypeptideversus a control antigen or epitope such as a polypeptide that is notthe particular antigen/epitope or polypeptide. Methods of preparingpolyclonal or monoclonal antibodies are well known to those skilled inthe art (see, for example, Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press (1988)). If desired, thepeptide antigens used to generate antibodies can be coupled to anappropriate carrier, as desired and described in Harlow and Lane, supra,1988.

In addition, the term “antibody” as used herein includes naturallyoccurring antibodies as well as non-naturally occurring antibodies,including, for example, single chain antibodies, chimeric, bifunctionaland humanized antibodies, as well as antigen-binding fragments thereof.Such non-naturally occurring antibodies can be constructed using solidphase peptide synthesis, can be produced recombinantly or can beobtained, for example, by screening combinatorial libraries consistingof variable heavy chains and variable light chains as described by Huseet al. (Science 246:1275-1281 (1989)). These and other methods of makingfunctional antibodies are well known to those skilled in the art (Winterand Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature341:544-546 (1989); Harlow and Lane, supra, 1988); Hilyard et al.,Protein Engineering: A practical approach (IRL Press 1992); Borrabeck,Antibody Engineering, 2d ed. (Oxford University Press 1995)).

The cell surface glycoprotein capture technology disclosed herein allowsfor specific detection and relative quantification via stable isotopelabeling of biologically and potentially medically relevant cell surfacemarker proteins, including those partitioning into lipid rafts (see, forexample, Wollscheid et al., Subcell. Biochem. 37:121-152 (2004);Wollscheid et al., Curr. Opin. Immunol. 16:337-344 (2004); Desiere etal., Genome Biol. 6:R9 (2004)). In contrast to previous approaches, thepresent method allows the identification of proteins in any oneexperiment that represent genuine plasma membrane molecules at the timeof the cell surface labeling. Thus, the present method allowsconfirmation and annotation of proteins in publicly available databasesas bona fide plasma membrane proteins. Furthermore, the initial resultsallow the specification of the experimentally verified N-glycosylationsite(s) within the identified proteins. For the first time, suchtechnology will allow for the scanning of cell surfaces in adiscovery-driven approach. Known as well as hypothetical proteinsinvolved in lipid raft and/or T cell signaling have already beenidentified, which can be followed up to more precisely determine theirbiological function.

The present methods allow transfer of the cell surface and glycoproteinlabeling strategy to target plasma membrane proteins in primary cells,for example, macrophages, stem cells, and the like, and tissue samplesfrom leukemia (CLL/ALL) and prostate cancer patients, for example, incollaboration with National Cancer Institute (NCI) and the FredHutchinson Cancer Research Center (FHCRC), as well as to other desiredcell or tissue samples. The identification of peptides can betransferred to the MALDI platform. Additional glycopolypeptide capturetechniques can also be used, for example, magnetic hydrazide microbeadsfor covalent attachment to cell surface glycoproteins as a alternateenrichment strategy. These beads allow identification not only ofN-linked glycopeptides, but also O-linked and GPI-linkedpeptides/proteins, further increasing coverage of the plasma membrane(PM) subproteome.

Although exemplified herein with a solid phase glycopolypeptide capturetechnology, for example using biocytin hydrazide, it is understood thatother glycopolypeptide capture methods can be used, such as those taughtin U.S. Publications 2004/0023306 and 2006/0141528, each of which isincorporated herein be reference. Other methods for identifyingglycosylation sites can utilize a solution based strategy that can beused to capture glycoproteins from solution. This solution can be thetissue culture media from cells in culture, blood or other bodilyfluids, or lysate of cells (see for example, Zhang et al., Nat.Biotechnol. 21:660-666 (2003), which is incorporated herein byreference. An exemplary method of purifying and identifyingglycoproteins from solution is described in Example III. It is thusunderstood that a variety of methods can be used to identify authenticglycosylation sites, in particular using mass spectrometry.

Thus, the methods of the invention can be used generate antibodiesagainst authentic glycosylation sites of a glycopolypeptide. The methodsare particularly useful for generating antibodies to authenticglycosylation sites identified by mass spectrometry. As exemplifiedherein, the glycosylation sites can be N-linked glycosylation sites, butother authentic glycosylation sites such as O-linked or GPI-linked sitescan be used. The methods are particularly useful for generatingantibodies against authentic N-linked glycosylation sites. Such N-linkedglycosylation sites can be identified, for example, using chemicalcapture such as biocytin hydrazide or other carbohydrate-specificchemistries, as described herein. Such methods allow for the enrichmentfor glycopolypeptides that can be subsequently analyzed by massspectrometry.

Additional studies can be performed using a metabolic labeling strategyin vivo with a modified sugar that gets incorporated into cell surfaceglycoproteins, similarly allowing for their selective isolation andcharacterization of glycoproteins. Further, the cell surfaceglycoprotein labeling strategy can be combined with quantitativeproteomics methods, such as those taught in U.S. Publications2004/0023306 and 2006/0141528. Other methods include usingamino-reactive isobaric tags such as iTRAQ™ reagents (AppliedBiosystems, Foster City Calif.), which can be employed to simultaneouslyidentify and provide relative quantitation of up to four related proteinsamples. This can be done to elucidate the role of newly identified keycell surface molecules in follow-up experiments as potentialdifferentiation or bio-markers, using, for example, green fluorescentprotein (GFP)-tagging, tandem affinity purification (TAP)-tagging,generation of antibodies, and the like.

The methods can also be used to investigate post-translationalmodifications in cell surface proteins and lipid rafts, for example,using solid phase technology allowing characterization of proteinphosphorylation sites in cell surface and lipid raft co-isolatingproteins via LC-MS/MS (see Tao et al., Nat. Methods 2:591-598 (2005)).

The above-described cell surface scanning technology has great potentialto cast new light on the membrane and lipid raft proteins involved insignal transduction. A combination of biology, chemistry, proteomics andbioinformatics has proven to be a powerful combination of tools for theidentification of new and unknown proteins relevant to the system understudy, as well as for the identification of known proteins whosesignaling involvement would not likely have been predicted from existingdata. With an ability to identify surface proteins at high confidence,this allows for the improved elucidation of differentiation markers fordisease and for the identification of better targets for immunotherapyin the case of cancer or other diseases. In addition, the methodologiesare widely applicable to most cell systems.

The present methods allow for cell surface scanning in adiscovery-driven approach, which was impossible before, to specificallyidentify cell surface proteins in a multiplexed fashion via MS. Thepresent methods also allow the determination of which proteins areexpressed on the cell surface of a given cell. The methods also allowfor biomarker discovery, for example, what differentiates a cancer cellfrom a “normal” cell. The methods additionally allow discovery ofgeneral differentiation markers, for example, for developmental stagesof cells, lineage commitment of stem cells, and the like. The methodsfurther allow identification of cell surface proteins, and the methodsrelating to identification of protein N-glycosylation sites allowsdetermination of the orientation of the identified protein within theplasma membrane.

The invention additionally provides a method to generate anantigen-specific antibody such as a peptide-specific antibody, forexample, a peptide containing a glycosylation site (see Example V). Themethod can include the steps of immunizing an animal with an antigensuch as a peptide to generate an immune response against the antigen,isolating antibody producing cells from the immunized animal, andscreening the isolated antibody producing cells for expression of anantibody against the immunizing antigen. A cell expressing a desiredantibody can be isolated, for example, using FACS and used to generate amonoclonal antibody using well known methods. If desired, more than oneantigen can be used to immunize the animal. Thus, the invention can beused to immunize with a plurality of antigens such as a plurality ofpeptide antigens, for example, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 ormore, 20 or more, or even greater, as desired, and isolated antibodyproducing cells can be screened for expression of one or more, andpotentially all of the immunizing antigens. Thus, the methods can beused to efficiently immunize a single animal with multiple antigens andidentify appropriate antibody producing cells such at that a pluralityof monoclonal antibodies can be efficiently produced against a pluralityof antigens with immunization of a single animal (see Example V).

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Cell Surface Scanning Protocol

This example describes a protocol for labeling and characterizing cellsurface glycopolypeptides.

MATERIALS, REAGENTS AND BUFFERS: Labeling buffer: phosphate bufferedsaline (PBS) pH6.5, 0.1% fetal bovine serum (FBS), the pH of the bufferis critical; Periodate, 5 g (#20504; Pierce;); Biocytin Hydrazide, 25 mg(#90060, Biotium;); Hypotonic lysis buffer: 10 mM Tris pH 7.5, 0.5 MMgCl₂; 15 ml Dounce Tissue Grinder (Wheaton;); Membrane prep buffer: 280mM sucrose, 50 mM Morpholinoethanesulfonic acid (MES) pH 6, 450 mM NaCl,10 mM MgCl₂; Ultacentrifuge: L8-M Ultra (Beckmann); Beckmann SW41 swingrotor, 6×12 ml tubes; SW41 rotor; 35,000 rpm, relative centrifugal force(rcf) 151,263; Ultra Clear Centrifuge tubes, 12 ml; (Beckmann #344059;UltraLink immobilized Streptavidin PLUS (Pierce; #53117); EndoproteinaseLys-C, Excision Grade, 5 μg (#324715, Calbiochem); sequencing grademodified Trypsin, 100 μg (#V5113, Promega); RapiGest (Waters); Ammoniumbicarbonate (Sigma; St. Louis Mo.); Peptide: N-Glycosidase F (PNGase F)(New England Biolabs).

HARVESTING CELLS: Collect cells (at least 5×10⁸) in a 50 mL Falcon tubeand wash 2× with 50 mL of labeling buffer.

PERIODATE STIMULATION: Dissolve 0.021 g Sodium-meta-periodate in 1 mL ofLabeling Buffer and add 500 μL of this solution to 40 mL of cellsolution (cell pellet suspended in labeling buffer). Rotate cells atroom temperature in the dark for 15 min. Then centrifuge cells for 5min. at 1500 rpm. Discard the supernatant and wash cells 2× in 50 mLlabeling buffer to remove residual periodate and to deplete deadcells/fragments.

CELL SURFACE LABELING: Resuspend pellet in a total volume of 11 mLlabeling buffer, then dissolve 30 mg of Biocytin hydrazide in 1 mLlabeling buffer. Add this to the cell solution. Label cells for 60 minat room temperature on a rotator on slow speed. Bring volume up to 50mL, invert several times, and spin at 1500 rpm for 5 min. Discard thesupernatant and wash cells 2× in 50 mL labeling buffer to removeresidual biocytin hydrazide and to deplete dead cells/fragments.

CELL LYSIS/MEMBRANE PREPARATION: Resuspend cells in 12 mL of detergentfree, hypotonic lysis buffer. Allow the cells to swell for 10 min on icebefore homogenization. Homogenize cells with 30 strokes using a Douncehomogenizer on ice. Transfer lysate to Eppendorf tubes and spin downnuclear debris at ˜2500×g at 4° C. for 10 min. Transfer supernatant to aFalcon tube, resuspend nuclear pellets in a total of 10 mL of lysisbuffer (take aliquot, 10 μl), and repeat membrane extraction, (takealiquots, 10 μl each), for a total of two combined membrane fractions.Add an equal volume of membrane prep buffer and allow to sit on ice for10 minutes. Centrifuge these membrane fractions at 35000 rpm for 1 hourat 4° C. Collect the membranes into a single tube using 200 μl ofammonium bicarbonate. Repeat with 100-200 μl to gather residualmembranes. Wash fractions with ˜200 μl membrane wash buffer (0.025 MNa₂CO₃ pH 11) and let sit on ice for 30 minutes. Spin samples at 35,000rpm for 30 minutes to remove all traces of Na₂CO₃. Gather pellet withammonium bicarbonate. Dissolve in 50 μl of Rapigest™ (10× stocksolution). Add 2-3 μl of tris(2-carboxyethyl)phosphine hydrochlorideTCEP depending on the amount of membrane collected. Allow membranes todissolve overnight at 4° C. Sonicate sample for 15 min to obtain a clearsolution. (take aliquot, 5 μl).

GLYCOPEPTIDE CAPTURE: Digest membranes for 4-6 hours with 1 μg (orsuitable amount) of Lys-C (take aliquot, 5 μl). Use 50 μl, of Lys-C androtate at 37° C. Subsequently, add 20 μg (or suitable amount) of trypsinand digest overnight at 37° C. (take aliquot, 5 μl). Inactivate LysC andtrypsin partially by heating the sample to 100° C. for 10 min. Allow thesample to cool down and add 10 μL protease inhibitor cocktail. Spinsample at 14,000 rpm for 10 min. Transfer supernatant into new tube.Discard the pellet, if any. Gather and assemble tubes for capture. Add 1mL of UltraLink Streptavidin PLUS beads to the column. Spin off anyexcess liquid after transferring beads to the column and add ammoniumbicarbonate to wash. Add digested peptides to column and mix gently bypipetting. Incubate beads with peptides for 1 hour in head over headshaker. Spin beads down. Save flow through for analysis ofnon-glycosylated peptides (Take aliquot; check for depletion ofbiotinylated molecules; Dot Blot). Wash beads with at least 10 mL 5MNaCl, followed by 10 mL StimLys buffer 0.5% Triton, followed by 10 mL 5MNaCl, followed by at least 10 mL 100 mM sodium carbonate pH11, followedby at least 5 mL 100 mM ammonium bicarbonate. Suspend the beads in 600μL 100 mM ammonium bicarbonate and elute glycoproteins from the beadsusing 1 μl PNGaseF overnight at 37 C. Spin off ammonium bicarbonate intoa fresh 2 mL tube. Repeat by adding additional ammonium bicarbonate andkeeping the flow through. This flow through contains the cleavedpeptides. Dry down eluates using a SpeedVac and suspend peptides in ˜40μl HPLC Buffer A for LCMS/MS or in a buffer suitable for furtherseparation. Transfer to small eppendorf tube and spin down at max. speedfor few minutes to collect any precipitate at the bottom. Repeat asneeded. Transfer to tube for MS analysis.

EXAMPLE II Chemical Tagging and Profiling of Plasma MembraneGlycoproteins by Mass Spectrometry

This example describes profiling of plasma membrane glycopolypeptides bychemical tagging and characterization by MS.

A biocytin-hydrazide based purification of membrane proteins isdescribed. Briefly, the purification of extracellular membrane proteinsusing biocytin-hydrazide uses five basic steps. (1) Gentle oxidation ofthe sugar molecules present on extracellular glycoproteins with sodiummeta-periodate converts the cis-diol groups of carbohydrates toaldehydes. (2) Biocytin hydrazide, which is membrane impermeable, isadded atop the live cells to form covalent hydrazone bonds withextracellular glycoproteins. (3) Cells are lysed, and a crude microsomalpreparation is made by ultra-centrifugation. (4) Proteins are digestedto peptides with trypsin. (5) Modified glycopeptides are captured usingstreptavidin-agarose. (6) The beads are washed and peptides are releasedfrom the resin by enzymatic cleavage with PNGase-F, which cleavesbetween the innermost GlcNAc and asparagine residues of high mannose,hybrid and complex oligosaccharides. This cleavage deaminates theasparagine residue to an aspartic acid residue, a change that can addspecificity to peptide identification. Steps one and two are illustratedin FIG. 1. This method has been developed that permits high efficiencypurification of membrane glycopeptides.

New methodologies for the specific labeling of proteins residing in theplasma membrane have been developed. One approach utilizes a three-steptandem affinity labeling strategy to confer this desired specificity forthe plasma membrane (PM) (FIG. 2) and lipid raft subproteomes (seeExample I). These steps are: i) gentle, covalent chemical labeling ofcarbohydrate-containing proteins on living cells; ii) specific enzymaticpeptide release that allows for systematic and selective identificationof N-glycosylated peptides derived from the surface glycoproteins; iii)subsequent peptide and protein identification via reversed phasecapillary liquid chromatography coupled to tandem mass spectrometry(LC-MS/MS). Results from model cell lines are yielding a never seenbefore degree of specificity for the detection of low abundance lipidraft-associated and cell surface molecules, with <10% contamination fromintracellular and nonglycosylated peptides/proteins (FIG. 3B). The“contaminations” can be singled out in a bioinformatics approach,yielding 100% bona fide plasma membrane proteins. A second, similarapproach is also under investigation, in which cells are metabolicallylabeled in vivo with a modified sugar that gets incorporated into cellsurface glycoproteins, again allowing for their selective isolation andcharacterization.

As shown in FIG. 2, the cell surface labeling technology is cell surfacespecific. Shown are Ramos B cells upon visualization of the tagged cellsurface proteins, which are subsequently identified via LC-MS/MS.

FIG. 3 shows identified proteins of lipid raft co-isolated proteinsbefore and after applying the cell surface glyco-capture technology.FIG. 3A shows identified proteins in a membrane preparation withoutusing the cell surface glycocapture technology. FIG. 3B shows identifiedproteins in a membrane preparation using the cell surface glyco-capturetechnology. Shown in each case is a partial list of proteinidentifications from a typical LC-MS/MS experiment, sorted according tothe total number of identified peptides, with a protein probability ofhigher than 0.5, as calculated by ProteinProphet™ using statisticalanalysis (Nesvizhskii, et al., Anal. Chem. 75:4646-4658 (2003)). Theprotein probability is an indicator for the likelihood that a particularprotein was present in the sample. Displayed columns include the IPIannotation; the number of uniquely identified peptides/protein; thetotal number of identified peptides/protein. The SOSUI webserver wasused to predict transmembrane domains in the retrieved proteinsequences. As shown in FIG. 3A, mostly soluble proteins were identifiedcompared to FIG. 3B, where only membrane proteins were identified.

For capture of membrane glycopeptides, a standard “membrane preparation”using hypotonic lysis and differential centrifugation typically producesan impure mixture of microsomes, contaminated with high abundanceintracellular cytoplasmic/structural proteins. A proteomic study of sucha preparation yields a diverse group of protein identifications withlimited specificity for the extracellular membrane. An experiment wasperformed using RAMOS B lymphocytes, which yielded a typical result. Themost abundant proteins from this experiment are not membrane specific,but abundant mitochondrial and ribosomal proteins. Few have membranespanning domains. A similar study investigating the contents of lipidrafts using differential centrifugation produced similar non-specificresults (von Haller et al., Proteomics, 1:1010-21 (2001)). In contrastusing the present method based on biocytin hydrazide, the proteins foundare highly enriched for bona fide membrane proteins as illustrated inFIG. 3B, where it is seen that all of the proteins identified have atleast one membrane spanning domain. In developing this method, specificwashing steps have been added that significantly reduce the non-specificbinding to streptavidin agarose, and typically find that approximatelyninety percent of proteins identified are membrane proteins. Additionalwash steps can be included, as desired. A typical mass spectrometryresult for a single glycoprotein is shown in FIG. 4.

EXAMPLE III Purification of Glycoproteins from Solution

This example describes the analysis of serum based on the selectiveextraction of glycosylated proteins using hydrazide resin, followed bydigestion with trypsin and elution of only glycopeptides withPeptide-N-Glycosidase F (PNGase-F). This method was also applied toLNCaP cells to improve the specificity for membrane or secretedproteins. This analysis identified 104 unique peptides mapping to 64unique proteins (1.6 peptides per protein) (Zhang et al., Nat.Biotechnol. 21:660-666 (2003)). All the peptides identified containedthe conserved N-linked glycosylation motif (N—X—S/T), indicating thatformerly N-glycosylated peptides were isolated with high selectivity.Using information from the SWISS-PROT database or the prediction toolPSORT II, 70% were found to be bona fide or predicted transmembraneproteins. The non-transmembrane proteins were mostly designated aseither extracellular, 11%, or lysosomal, 14%, thus belonging to twocellular compartments also known to be enriched for glycoproteins.

EXAMPLE IV Generation of a List of T-Lymphocyte Enriched ProteinCandidates for Analysis

A current dataset contains approximately 250 membrane proteins fromRamos B cells and about the same number from Jurkat T cells. This listwas obtained by pooling the multiple experiments on these cell linesused during method development. The overlap between the B and T celllines using this method is approximately 35%. This does not mean thatproteins seen on one line are not present on the other; expression levelfor proteins could be low enough that they fall below the limits ofdetection. A more formal quantitative study is needed to determinerelative protein expression. The list represents an approximation. Table1 details 35 proteins which will be used as candidates because they areoverrepresented on Jurkat T cells. Table 1 lists a sample of 35 proteins(SEQ ID NOS:1-35) found to be overrepresented on Jurkat T cells comparedto RAMOS B cells using the surface glycocapture technique. A singlepeptide sequence containing the site of glycosylation (in bold) isgiven, though many of these proteins have multiple glycosylation sitesidentified. The list consists of GPI anchored proteins (indicated with“*”), and proteins with variable numbers of transmembrane domains,indicated in the tm column. The SOSUI membrane spanning information isalso indicated.

TABLE 1 Sample of Proteins Overrepresented on Jurkat T Cells Comparedto RAMOS B Cells. UP SYMBOL DESCRIPTION PEPTIDE SEQUENCE SOSUI TMTHY1_HUMAN Thy-1 cell surface HENTSSSPIQYEFSLTR M2* 0 antigen variant(SEQ ID NO: 1) GFRAl_HUMAN Splice Isoform 1 of ETNFSLASGLEAK M1* 0GDNF family (SEQ ID NO: 2) receptor alpha-1 CD48_HUMAN CD48 antigenELQNSVLETTLMPHNYSR M2* 1 precursor (SEQ ID NO: 3) LFA3_HUMANCD 58 Lymphocyte VYLDTVSGSLTIYNLTSSDEDEYE M1* 1 function-associatedMESPNITDTMK antigen 3 (SEQ ID NO: 4) IGF1R_HUMAN Insulin-like growthLILGEEQLEGNYSFYVLDNQNLQQ M2 1 factor 1 receptor LWDWDHR precursor(SEQ ID NO: 5) CD1D_HUMAN T-cell surface TDGLAWLGELQTHSWSNDSDTVR M2 1glycoprotein CD 1 d (SEQ ID NO: 6) precursor BST2_HUMAN Bone marrowNVTHLLQQELTEAQK M2 1 stromal antigen 2 (SEQ ID NO: 7) CD7_HUMANT-cell antigen CD7 DFSGSQDNLTITMHR M2 1 precursor (SEQ ID NO: 8)CD166_HUMAN CD166 antigen TVNSLNVSAISIPEHDEADEISDEN M2 1 precursor R(SEQ ID NO: 9) MUC18_HUMAN Isoform 1 of Cell PTISWNVNGTASEQDQDPQR M2 1surface glycoprotein (SEQ ID NO: 10) MUC18 ICAM2_HUMAN IntercellularAPQEATATFNSTADR M2 1 adhesion molecule (SEQ ID NO: 11) 2 precursorADA10_HUMAN ADAM 10 INTTADEKDPTNPFR M2 1 precursor (SEQ ID NO: 12)CD1C_HUMAN CD1C antigen, GNFSNEELSDLELLFR M2 1 c polypeptide(SEQ ID NO: 13) CD69_HUMAN Early activation EFNNWFNVTGSDK M1 1antigen CD69 (SEQ ID NO: 14) O95266_HUMAN LeukocyteITQSLMASVNSTCNVTLTCSVEK M2 1 differentiation (SEQ ID NO: 15)antigen CD84 isoform OXRP_HUMAN 150 kDa oxygen- VFGSQNLTTVK M1 1regulated protein (SEQ ID NO: 16) precursor EMB_HUMAN Embigin precursorCQNCFPLNWTWYSSNGSVK M1 1 (SEQ ID NO: 17) EFNB1_HUMAN Ephrin-B1HHDYYITSTSNGSLEGLENR M2 1 precursor (SEQ ID NO: 18) SLAF6_HUMANactivating NK NIQVTNHSQLFQNMTCELHLTCS M2 1 receptor precursorVEDADDNVSFR (SEQ ID NO: 19) NEO1_HUMAN Splice Isoform 1 ofVAALTINGTGPATDWLSAETFESD M2 1 Neogenin precursor LDETR (SEQ ID NO: 20)LYAM1_HUMAN L-selectin precursor DNYTDLVAIQNK M1 1 (SEQ ID NO: 21)NICA_HUMAN Splice Isoform 2 of DLYEYSWVQGPLHSNETDR S 1Nicastrin precursor (SEQ ID NO: 22) SORT_HUMAN Sortilin precursorDITDLINNTFIR M2 1 (SEQ ID NO: 23) CD45_HUMAN Protein tyrosineYHLEVEAGNTLVRNESHK M2 2 phosphatase, (SEQ ID NO: 24) receptor type, CPVR1_HUMAN Poliovirus receptor- SGQVEVNITEFPYTPSPPEHGR M2 2related protein 1 (SEQ ID NO: 25) precursor SEM4D_HUMAN Semaphorin-4DEAVFAVNALNISEK M2 2 precursor (SEQ ID NO: 26) PTK7_HUMAN PTK7 proteinQDVNITVATVPSWLK M2 2 tyrosine kinase 7 (SEQ ID NO: 27) isoformCD82_HUMAN CD82 antigen, DYNSSREDSLQDAWDYVQAQVK M4 4 Kangai 1(SEQ ID NO: 28) CD151_HUMAN CD151 antigen YHQPGHEAVTSAVDQLQQEFHCC M3 4GSNNSQDWR (SEQ ID NO: 29) TSN4_HUMAN Tetraspanin-4 CCGVSNYTDWFEVYNATR M44 (SEQ ID NO: 30) LASS2_HUMAN LAG1 longevity LWLPVNLTWADLEDR M5 5assurance (SEQ ID NO: 31) homolog 2 KCAC3_HUMAN Potassium voltage-DYPASTSQDSFEAAGNSTSGSR M5 5 gated channel (SEQ ID NO: 32) LPHN1_HUMANSplice Isoform 1 of DMNATEQVHTATMLLDVLEEGA M8 8 Latrophilin-1 FLLADNVRprecursor (SEQ ID NO: 33) GTR1_HUMAN facilitated glucose VIEEFYNQTWVHRM12 12 transporter (SEQ ID NO: 34) member 1 CTR1_HUMAN High-affinityNWQLTEEDFGNTSGR M14 14 cationic amino acid (SEQ ID NO: 35) transporter 1

EXAMPLE V A Method for High Through-Put Antibody Production UsingGlycoprotein Sequences Conjugated to Sulfhydryl and Analyzed by Facs

Introduction

The ability to identify sensitive and specific diagnostic markercandidates from proteins to monitor health and disease progression is asignificant problem in the field of diagnostics. Currently,identification of candidate proteins, development of prototypes(including antibody development) and initial validation of antibodiesfor diagnostics using standard techniques is slow, requires arecombinant expression system, and purification requires many steps. Insome diseases, (i.e., cancer), the specificity of the antibody is testedin different cancer tissues with differing specificity, sensitivity andsuccess. (see Zolg and Langen, Mol. Cell. Proteomics 3:345-354 (2004)).

In this example, a high through-put method is described to generatediagnostic marker candidates to develop antibodies against specificepitopes (portion of a molecule to which an antibody binds) tosignificantly increase specificity, sensitivity and decrease developmenttime. Although polyclonal antibodies can be produced, the ultimate goalis to develop monoclonal antibodies and the methods described hereinprovide such antibodies. Methods described above will be used toidentify and sequence proteins in the native configuration. The proteinsequence identified will be used to generate peptides for antibodydevelopment. The method is tested in a cancer disease model using thehigh through-put testing model described.

Materials

Exemplary materials such as Keyhole limpet hemocyanin (KLH) will besupplied as a lyophilized powder, or other suitable form (available, forexample, from Cal Bio Chem., La Jolla, Calif.). Alternatively, albuminmay be used for conjugation or any other material or substance known inthe art may be used. Fluorescein-5-maleimide will be supplied by Pierce(Rockford, Ill.). Alternatively, any other fluorophore known in the artmay be used. Peptides to identified protein sequences will be generatedby a commercial source such as Sigma-Aldrich (St. Louis Mo.).

Method

Using the methods described herein, protein glycosylation sites will beidentified. Sites proximal to these glycosylation sites as describedwill be chosen for immunization. In one method, specific cysteineresidues will be used for labeling. Identified protein sequences that donot have a cysteine residue will have a cysteine residue incorporated atthe end of the identified protein sequence to use maleimide labeling,which reacts with the sulfur moiety in cysteine. Maleimide is intendedto be exemplary and any other type of labeling known to the skilledartisan is expected to produce the same results. The protein sequencewith the added cysteine residue will be used to generate a syntheticpeptide using a commercial vendor. The resulting peptide will then beconjugated to keyhole limpet hemocyanin (KLH) as previously describedand injected into an animal. KLH is intended to be exemplary. Any otherprotein (i.e, albumin) that may be used for coupling can be used toproduce the desired results. For the proposed method, an exemplaryanimal is a rabbit, but any suitable animal may be used to produceantibodies. The synthetic peptide will also be coupled tofluorescein-5-maleimide or any other fluorophore known in the art. Thisexemplary reagent will be used to identify antibody producing cells inthe immunized animal. The fluorescein labeled peptide is incubated witha preparation of antibody producing cells from the immunized animal tolabel only those cells with specific reactivity to the immunizingpeptide. Using Fluorescence Activated Cell Sorting (FACS), cells withthe fluorescein signal will be identified and only those cells withfluorescein signal will be collected for further testing.

Test Animals

Test animals will be used to generate antibodies. Exemplary test animalsinclude, rabbits, rats or mice or any other animal that would generatean immune response. Animals will be purchased from a certified vendor.Food and water will be provided ad libitum. Test animals will be allowedto acclimate in a temperature and humidity controlled environment forapproximately one week prior to the commencement of experimentalprocedures.

EXAMPLE Proposed Cancer Diagnostic Markers

In one experiment, the methods described will be used to generateantibodies to ErbB-2, a membrane protein marker used as a diagnosticprotein in breast cancer (see: Roskoski, R., Biochem Biophys Res Comm(2004) 319:1-11). This protein has been identified in UniPep(db.systemsbiology.net/sbeams/cgi/Glycopeptide/Glyco_prediction.cgi?action=Show_detail_form&ipi_data_id=19935)with unique glycosylation sites.

Some of these Sites are:

NXS/T location Sequence  68 R.HLYQGCQVVQGNLELTYLPTNASLS.F(SEQ ID NO: 36) 124 R.GTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLR.E(SEQ ID NO: 37) (unknown)

Eight peptides (for each identified glycosylation site) will begenerated wherein the cysteine residue is conjugated to KLH. Two rabbitswill be injected with unique peptides conjugated to KU-I correspondingto 2 glycosylation sites. After the immune response is generated, memoryB or plasma cells will be obtained from the animals and will be sortedby FACS for reactivity to one of the fluorescent peptides. Each antibodyproducing clonal population will be tested using an indirect ELISA assayor surface plasmon resonance to determine the specific peptide againstwhich it is reactive.

The cell lines reactive to the fluorescein tagged peptides used forimmunization are tested for reactivity to ErbB-2 to breast cancer celllines such as MCF-7, T-47D, SKBR3, MDA-MB-435, and MDA-MB-231 by Westernblot, immunohistochemistry and immunoprecipitation of ErbB-2. Eachreaction will be quantified and compared to known standards as antibodyresponses to ErbB-2 are widely known. It is anticipated that Erb B-2 isexemplary and other proteins may serve as useful biomarkers. It isexpected that the tagged peptides will produce antibodies to nativeproteins that will have a statistically greater reactivity when testedby standard immunology methods as the peptides generated will reflectthe native protein sequence since they are proximal to a glycosylationsite.

Results

The method described herein does not require a recombinant expressionsystem to produce an immunogen, which will decrease the possibility ofprotein folding and glycosylation, both of which diminish specificantibody responses to the native protein. By identifying proteins basedon specific glycosylation sites, the opportunity to produce antibodiesto native proteins is enhanced.

An antibody will be developed using peptide immunization based on apredetermined protein sequence that will be specifically identifiedfollowed by an antibody response that is monitored by a fluorescentmarker that is tagged to the identical protein sequence. The methodsdescribed herein are highly specific and designed to sequence a nativeprotein, tag it with fluorescence and identify the antibody response.The method described herein allows immunization of a single animal withmultiple peptides simultaneously, with reactivity to be determined afterclonal populations are defined, improving the efficiency of theprocedure.

This proposed method will significantly decrease the steps ofdevelopment and will potentially provide greater antibody specificityand significantly decrease development time and costs. In addition, theproduct generated will be a monoclonal antibody rather than a polyclonalantibody. This is highly desirable when downstream applications includediagnostic or therapeutic assays, because reagent purity and stabilityof reactivity is essential for any biologic assay or therapeuticreagent.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

1. A method to produce an immunizing peptide for generating an antibodyspecific for the glycosylated form of a glycopolypeptide, comprising:(a) isolating a glycosylated portion of said glycopolypeptide; (b)identifying by mass spectroscopy an amino acid sequence consisting of6-40 amino acids whose C-terminus or N-terminus is within 5 amino acidsupstream or downstream respectively of the N-terminus or C-terminusrespectively of a glycosylated site of said glycosylated portion ofglycopolypeptide obtained in step (a), wherein said site has thesequence N-X-S/T; (c) producing said immunizing peptide corresponding tothe amino acid sequence identified in step (b).
 2. The method of claim1, wherein the glycosylated portion is isolated by a method includingthe step of treating said glycopolypeptide with periodate, followed bytreating with biocytin hydrazine, and coupling the resultant tostreptavidin.
 3. The method of claim 1, wherein the glycosylated portionis isolated by a method including the step of treating said glycopeptidewith periodate followed by treating with hydrazide resin.
 4. The methodof claim 1, wherein said peptide consists of 6-25 amino acids.
 5. Themethod of claim 1, wherein said peptide consists of 6-15 amino acids. 6.The method of claim 1, wherein said peptide consists of 6-10 aminoacids.
 7. The method of claim 1, wherein said peptide consists of 6-8amino acids.
 8. The method of claim 1, which further comprises couplingto a heterologous peptide.
 9. The method of claim 8, wherein theheterologous peptide is keyhole limpet hemocyanin (KLH).